EPITOPE MAPPING OF A SERIES OF HUMAN THYMIDYLATE SYNTHASE MONOCLONAL-ANTIBODIES

We have reported previously the development and application of several monoclonal antibodies to thymidylate synthase (TS). In this study, we used a series of overlapping 17-mer peptides that spanned the entire TS protein to map the epitope recognized bg three TS monoclonal antibo dies (TS 106, TS 109, and TS 110). Using an ELISA, we identified two p eptides (R-126-F-142 and L-131-R-147) that bound all three antibodies, which suggests that each antibody recognized a similar epitope on TS. A second set of peptides, representing sequential single-residue trun cations from either the amino terminus or the carboxyl terminus starti ng with a G(129)-E-145 17-mer, was synthesized. A 10-amino acid sequen ce P-133-F-142 (PVYGFQWRHF) was identified as the binding epitope for all three antibodies. Further investigation via substitution mutationa l analysis of each residue within this epitope revealed that residues F-137, W-139, R-140, H-141, and F-142 mere critical for maximal bindin g of TS 106 and TS 110. TS 109 showed a similar pattern except in rega rd to R-140, with which there mas no apparent loss of binding. In addi tion to the utility of the three antibodies in detecting and measuring TS levels, identification of the binding locus permits the potential application of these antibodies in the investigation of TS enzymatic a nd regulatory function.